Epigenetic erosion of H4K20me1 induced by inflammation drives aged stem cell ferroptosis

Aging is associated with a decline in stem cell functionality and number across the organism. In this study, we aimed to further unravel Muscle Stem Cells (MuSCs) aging by assessing how systemic factors influence MuSC fate decisions through long-term epigenetic landscape remodelling. As aging is intricately linked to a pro-inflammatory shift, we studied the epigenetic effects of inflammatory signals in MuSCs and measured decreased H4K20me1 levels. This loss disrupts MuSC quiescence, largely through epigenetic silencing of Notch target genes. In the setting of inflammatory signals or aging, the lack of Kmt5a and the subsequent absence of de novoH4K20me1 culminate in cell death by ferroptosis. Aged MuSCs manifest abnormal iron metabolism and reduced Gpx4 levels, resulting in the accumulation of intracellular iron, increased reactive oxygen species, genomic instability, and lipid peroxidation. We showed that ferroptosis is the predominant mode of cell death in aged MuSCs, with remarkably high levels of lipid peroxidation; a phenomenon we also observed in aged hematopoietic stem cells. Implementing preventative strategies to inhibit systemic inflammation prevented aged MuSC ferroptosis, preserving their numbers and regenerative capabilities. This intervention significantly enhanced aged muscle regeneration and strength recovery and extended both lifespan and healthspan in mice. This study delineates a previously underappreciated fate trajectory for stem cell aging, and offers meaningful insights into the treatment of age-related disorders.


Introduction
Aging is a complex biological process characterized by intricate cellular changes, central to which adult stem cells are crucial for maintaining tissue homeostasis.With age, the functionality of these stem cells markedly declines, leading to a plethora of physiological alterations.Aged skeletal muscles are plagued by decreased muscle mass and function and impaired regenerative capacity, which correlates with the loss of muscle stem cells (MuSCs) and increased in ammation [1][2][3] .Although the relationship between these events has been extensively studied, the molecular mechanisms underlying them remain unclear.
A core concept of aging is the loss of epigenetic information, with erosion of histone modi cations serving as one of its central axes 4,5 .Histone modi cations have been scarcely studied in MuSCs but have been identi ed as critical determinants of MuSC quiescence and activation 6,7 .Although previous studies have highlighted the role of H4K20 in adult stem cell function, the exact contribution of H4K20 during the aging process remains unknown 8,9 .In addition, identifying the mechanisms underlying epigenetic erosion remains a challenge in the study of stem cell aging.Growing evidence indicates the contribution of several extrinsic factors such as in ammation 10 .
With age, MuSCs lose quiescence and transition toward irreversible fates, such as death or senescence, leading to defective muscle repair 1,[11][12][13] .In the wake of the rst rejuvenation experiments using heteroparabiosis 14,15 , few studies have investigated how systemic molecular and cellular changes might affect stem cell rejuvenation, focusing instead on the stem cell niche, thus leaving a knowledge gap.In this study, we aimed to further unravel the mysteries of MuSC aging by assessing how systemic factors in uence MuSC fate decisions through long-term epigenetic landscape remodelling.By employing a comprehensive approach combining epigenomics, single-cell measurements, and functional testing of MuSCs from young and old mice, we investigated the molecular mechanisms underlying age-related defects in the fate, functionality, and survival of MuSCs.We present evidence that age-associated in ammation induces profound epigenetic remodeling in MuSCs, culminating in their premature exit from quiescence and susceptibility to an iron-dependent form of cell death termed ferroptosis.Our data underscores that acute systemic in ammation distinctly targets histone H4 lysine 20 methylation, subtly recon guring the MuSC transcriptome to a state that anticipates subsequent injury responses.During aging, chronic low-grade systemic in ammation results in compromised activity of lysine methyltransferase 5a (Kmt5a), thereby disrupting the crucial transcriptional programs necessary for preserving MuSC quiescence and survival.

Age-associated systemic in ammation and MuSCs epigenetic remodelling
To better understand the intricate relationship between systemic aging and MuSCs, we conducted a comprehensive analysis of the molecular and cellular characteristics of the murine aging circulatory system and compared them with both aged MuSC intrinsic changes and aged MuSC niche extrinsic changes (Figure 1A).First, we used whole blood counts followed by plasma collection to analyze the molecular and cellular blood pro les of young and aged mice.To ensure an unbiased approach, we rst screened over 100 cytokines in young and aged mice using a proteome pro ler array, followed by secondary validation of the identi ed candidates using quantitative multiplexing immunoassays.As expected, the plasma from aged mice displayed elevated levels of circulatory cytokines (TNFa, IL1a, IL1b IL-4, IL-6, CCL2, CCL7, CCL11, and CCL12) and myeloid cells, and a decline in lymphoid cells (Figure 1B, 1C, and Table S1).Together, these data support the widely recognized age-associated chronic low-grade in ammation, often referred to as "in ammaging," and myeloid bias resulting from the aging hematopoietic lineage.Secondly, when we interrogated the aged skeletal muscle transcriptome using our previously published dataset 16 , we found that the top three enriched activated pathway (Z-score > 2) were "Chemokine signaling", "Cytokine-Cytokine Interaction" and "Complement & Coagulation Cascades" (Table S1).This led us to wonder whether increased circulatory cytokine levels could affect aged skeletal muscle.Using a suite of computational analyses, we integrated our proteomics data with transcriptomic results to generate a ligand-receptor predictive model and the subsequent signaling activity 17 .We identi ed ve signaling predicted to be activated by our ligand-receptor model, including "Chemokine Signaling", "IL-7 signaling" and "TLR-signaling" (Figure 1D and E and Table S1).Interaction analysis predicted CCR2 to have the highest activation score, signi cantly contributing to all but one of the signaling pathways (Figure 1E).
We previously established that CCR2 signaling activation is a hallmark of skeletal muscle aging 1 .The levels of circulatory CCR2-ligands have also been thoroughly characterized as a measure of biological age, frailty, and age-associated systemic in ammation in both humans and mice 11,[18][19][20] .Building on this foundation, our rst objective was to elucidate the connection between systemic in ammation and the intrinsic changes observed in skeletal muscles and MuSCs with advancing age.We rst simulated a directed CCR2-mediated in ammatory response by systemically injecting recombinant CCL2, CCL7, and CCL8 into young animals as previously described 1 .Following injection, we recorded a transient in ammatory response, as evidenced by the increase in both the number of monocytes and the levels of pro-in ammatory cytokines in the blood of the treated mice (Figure S1E-J).We also assessed how this acute in ammatory response affects MuSCs, using CCR2-null mice as a negative control 1 .Although we observed no change in the raw number of MuSCs (Pax7+), stem cells from mice injected with chemokines displayed enhanced activation (Ki67+), cell cycling (EdU+), and accelerated myogenesis (MyoD/MyoG+) (Figure S1A-D).These have been previously described as features of the G alert state 21,22 .
Interestingly, these enhanced characteristics remained long after chemokine levels returned to baseline (Figure S1K-M), whereby we observed accelerated regeneration (Figure S1N-P), in line with previous reports regarding G alert .MuSCs that have experienced prior injury have a long-term enhanced regenerative capacity 23 , yet the mechanisms underlying this phenomenon are not well understood.These long-term functional changes prompted us to believe that MuSCs can adapt to acute in ammatory signals, which are indicative of epigenetic reprogramming 24,25 .
We hypothesized that age-associated systemic in ammation might affect the epigenome and transcriptome of MuSCs.Following our results, we performed an in-depth examination of the aged MuSC transcriptome and found that one of the most signi cantly enriched pathways was "Chromatin organization" (Figure 1F), in with our initial hypothesis and previous reports 7,26 .Of particular interest was the lysine methyltransferase Kmt5a, because of its crucial role in maintaining H4K20 methylation.Kmt5amediated catalysis of H4K20 monomethylation is required for subsequent di-and trimethylation, which is necessary for establishing constitutive heterochromatin.The dynamic regulation of both Kmt5a and H4K20me1 is also indispensable for proper cell cycle progression, which is intriguing given that MuSCs predominantly exist in a state of quiescence and aged MuSCs often die upon activation.We con rmed our RNA-seq results to show that both the Kmt5a gene and protein decreased in aged MuSCs (Figure 1I and J).Loss of Kmt5a was functionally re ected by decreased methylation of its main substrate, H4K20me1 (Figure G-M).Single-cell analysis revealed a shift in H4K20me1 intensity in the aged MuSC population.
Next, we assessed whether CCR2 activity is linked to Kmt5a repression.In response to the systemic delivery of CCR2-ligands, Kmt5a, but no other epigenetic genes, was signi cantly repressed in MuSCs (Figure S1Q and R).It was also accompanied by long-term transcriptional changes in both myogenic and cell cycle genes (Figure S1S and S1T).Notably, we observed the upregulation of activation genes (Myf5 and Dek) and downregulation of quiescence genes (Hes1 and Hey1).We found that MuSCs displayed long-term erosion of H4K20me1 and a sustained increase in MyoD+ cells lasting for at least six-weeks post-injection (Figure S1U-X).Single-cell analysis also pointed that CCR2-ligands treatment promoting a shift in H4K20me1 intensity, with the population of MuSCs derived from treated animals displaying lower H4K20me1 levels (Figure S1W and S1X).These results suggest that CCR2-mediated in ammation may trigger long-term epigenetic remodelling in MuSCs, which is potentially mediated by Kmt5a and H4K20 methylation.Thus, we further investigated the role of Kmt5a in MuSCs to better understand its effect on muscle aging.
Kmt5a is required for quiescence maintenance and MuSC survival after activation.
To assess the role of Kmt5a in quiescent MuSCs, we generated inducible Kmt5a MuSC-speci c knockout mice (Pax7 CreERT2/+ ; Kmt5a / [Kmt5a KO ]) (Figure S2A).Immediately following the deletion of Kmt5a, we did not observe a change in MuSC number in vivo, despite the vast majority of Kmt5a KO MuSCs lacking Kmt5a (>95% e ciency; orange arrows) (Figure S2B-D).Next, we cultured both wildtype (Pax7 +/+ ; Kmt5a / ) and Kmt5a KO mice to assess myogenic potential, cell proliferation, and survival.Freshly isolated MuSCs (Pax7+) were stained with myogenic markers to assess myogenesis progression toward commitment (MyoD) and terminal differentiation (MyoG) over time 1 .Ki67 and EdU were used to assess cell cycle re-entry (activation) and active cell proliferation, respectively.When we challenged the cells outside their niche, cultured Kmt5a-null cells displayed enhanced activation (Pax7+Ki67+), which directly correlated with the loss of H4K20me1 as early as 24hours after plating (Figure S2E-I).However, once MuSCs started actively dividing (EdU+) after 72h of culture, we observed a near-complete loss of H4K20me1, which is in line with the cell cycle regulation of H4K20me1.This decrease was accompanied by impaired myogenic terminal differentiation and cell expansion (Figure S2E-K).Further analyses revealed that Kmt5a KO MuSCs displayed aberrant morphological phenotypes, such as blebbing, pycnotic, and multiple nuclei, and a molecular signature suggestive of genomic instability (Figure S2L, M; red arrows).Molecular analysis of 72h cultured MuSCs supported this premise, as mutant cells displayed elevated y-H2AX and serine 15 phosphorylation of P53.
When challenged by acute sterile injury, the contribution of Kmt5a KO MuSCs to regeneration was disrupted, resulting in the apparent absence of muscle regeneration, as we did not observe centrally nucleated bers (CNF) among the injured mutants (Figure 2A).We also noted a macroscopic decrease in injured muscle size 21-day post-injury (dpi) (Figure S3A, B).To better index regeneration, we used embryonic myosin heavy chain (eMHC), a marker of immature (regenerating) skeletal muscle bers, throughout the regeneration process (Figure S4A).As expected, wild-type regenerating bers were nearly all positive for eMHC at early regeneration time points (4-and 7dpi), followed by the generation of large centrally nucleated bers (CNF), a hallmark of regeneration.In contrast, we did not observe any regenerating bers in Kmt5a KO mice past 7dpi (Figure S4B).By 60dpi, most of the injured area appeared to be reduced to remnants of the ECM and mononucleated cells.Additionally, virtually no surviving MuSCs were observed after regeneration (Figure 2B and S4C).When we investigated the fate of these MuSCs and their derived progenitors, we found a sharp decline in the number of myoblasts (MyoD+) in the mutant at 4dpi (Figure S4D-G).The number of terminally committed progenitors (MyoG+) brie y increased at 4dpi before virtually disappearing at 7dpi (Figure S4E-F).Next, we aimed to further understand the fate of Kmt5a KO MuSCs in response to direct and indirect environmental pressures.Since MuSCs and derived progenitors were lost between 4-and 7dpi, we assessed proliferative and myogenic capacity at 4dpi in both injured and uninjured contralateral (CL) limbs, where MuSCs are expected to enter the G alert state (Figure S4A) 21 .While MuSC numbers in Kmt5a KO mice rapidly declined in both injured and CL muscles (Figure S4C, S4I, and S4K), MuSCs derived from the injured limb displayed primed features reminiscent of G alert , including swifter entry into the cell cycle (Figure S4J and S4L), accelerated myogenic progression (Figure S4E-G), and high p-S6 levels compared to wild-type MuSCs (Figure S4M-O) 22 .
Previous reports have shown that H4K20me1 levels are regulated in a cell cycle-dependent manner in some somatic cells 27 .Based on our observations, H4K20me1 levels in quiescent MuSCs were relatively stable and fell mostly after the rst cell division, which occurs approximately 60h post-activation 21 (Figure S2E and S2O, P).We hypothesized that long-term loss of Kmt5a would eventually disrupt H4K20me1 maintenance by preventing de novo deposition of H4K20me1 and potentially altering the capacity of MuSCs to remain quiescent.To test this theory, we deleted Kmt5a and waited for 1, 3, or 6 weeks before assessing MuSC number and function in vivo (Figure 2C-O).Over time, the MuSC number progressively declined in a stochastic manner (Figure 2D).While MuSCs were nearly undetectable after six weeks, the few remaining MuSCs still displayed detectable levels of H4K20me1 (Figure S2O, P).Taken together, these results support our theory that the loss of H4K20me1 over time following Ktmt5a deletion does not occur immediately and simultaneously in every cell.Notably, from three weeks and onward, Kmt5a KO MuSCs displayed features of quiescence exit, depicted by elevated levels of pS6 (Figure 2E) and a higher number of Ki67-positive cells (Figure 2F,G), but a low number of actively cycling cells (EdU+) (Figure 2F, H).Next, we investigated the fate of the Kmt5a KO MuSCs in vivo.A previous report has demonstrated that tempering with H4K20me2 leads to spontaneous and precocious differentiation of MuSCs during homeostasis 9 .However, we did not nd MyoG+ cells in our mutants in homeostasis (Figure 1I).Since we observed a decline in survival and genomic instability in cultured Kmt5a KO MuSCs, which correlated with the loss of H4K20me1 (Figure S2E-N), we tested the possibility that Kmt5a KO MuSCs exited quiescence and entered cell death.We found a signi cant portion of mutant cells showing signs of cell death (~28% TUNEL+) (Figure 1J), together with an increased DNA damage response depicted by increased yH2AX and p53 signaling (Figure 1J, K).
Taken together, these results suggest that H4K20me1 regulation is an important factor in ne-tuning MuSC quiescence and, consequently, ensuring MuSC survival in response to extrinsic cues.Kmt5a maintains MuSC quiescence by epigenetically regulating Notch signaling through promoter-proximal pausing.
To identify the mechanisms by which Kmt5a affects MuSC quiescence maintenance, we conducted a suite of transcriptomic assays.Because the Kmt5a KO MuSC number declined stochastically over time (Figure 2D), we used single-cell RNA sequencing to assess the dynamic transcriptional changes in Kmt5anull MuSC fate over time.Unbiased clustering and UMAP reduction showed that wild-type cells were signi cantly different from the Kmt5a KO MuSCs.As expected, the Kmt5a KO MuSC population 2-weeks post deletion displayed a different transcriptomic pro le than the 6-weeks post deletion MuSCs, which surprisingly resembled 2-years knockout MuSCs (Figure 3A).Pseudo-time analysis show that a small subset of MuSCs exist only 2-weeks after Kmt5a deletion and present upregulation of myogenic differentiation genes Myog and Mymk, along with the cell cycle arrest gene Cdkn1c (Figure 3B, and Figure S5C-E).In every mutant sample, we observed a drift in the Kmt5a KO MuSC population, with large changes in quiescence regulatory genes, such as Notch Signaling 28 , Mitophagy 22 , and ECM-receptor interactions 29 (Figure S5A).Within this population, pseudo-time analysis showed that several key Notch-related genes and downstream targets (Notch, Jag1, Numb, and Rbpj) were downregulated over time, along with other myogenic markers (Pax7, Sdc4, and Spry1), consistent with the loss of quiescence (Figure S5C and E).
Next, we combined Bulk RNA-seq with PRO-seq to compare wild-type and Kmt5a KO MuSCs.RNA-seq was used as a measure of total mRNA, whereas PRO-seq was used to determine dynamic transcriptional changes at base-pair resolution 30 .Most of the signi cantly altered genes within the Notch signaling pathway were regulated through promoter-proximal pausing (Figure 3C).The pausing index signi cantly increased for several key Notch effectors (e.g., Rbpj and Dll1) and downstream targets (e.g., HeyL, Hes6, Dtx4, and Snw1), which correlated with signi cant gene repression.We also found the opposite to be true, as a decrease in the pausing index at Dll4 and Jag2 promoters, both Notch1 ligands that are usually repressed, correlated with increased expression 31 .Overall, Kmt5a deletion accounted for >80% of the altered Notch pathway gene expression, highlighting Kmt5a as a potential master transcriptional regulator of Notch signaling in quiescent MuSCs.This translated into lower levels of Rbp-jκ protein, a critical regulator of MuSC quiescence 32 (Figure 3D).Genetic deletion of Kmt5a in MuSCs did not immediately result in the repression of Notch genes (Figure S6A) despite the loss of detectable Kmt5a binding at the Rbpj TSS (Figure S6B).Instead, Notch target genes repression was observed at the earliest timepoint in our scRNA analysis and onward (Figure S5D and S5E).This was in line with the progressive loss of MuSC quiescence observed in the mutants during homeostasis (Figure 2D).Therefore, we wondered whether Kmt5a-mediated regulation of Notch genes was catalytically dependent.Since H4K20me1 was previously shown to promote transcription through RNA Polymerase II release during promoter-proximal pausing, we tested whether loss of H4K20me1 might be a necessary step for Notch genes repression 33 .We used a well-characterized selective Kmt5a inhibitor 34 that resulted in rapid H4K20me1 decline without affecting Kmt5a levels (Figure 3E, F).Upon catalytic inhibition of Kmt5a in MuSCs, the transcript levels of Hes1, Hey1, Notch1, and Rbpj were signi cantly decreased without affecting myogenesis genes (Figure 3G).We concluded that Kmt5a is an epigenetic regulator of Notch genes transcription.
Based on these results, we hypothesized that restoring Notch target gene expression could rescue Kmt5amediated loss of quiescence.To test this hypothesis, we crossed MuSC-speci c Kmt5a KO mice with the ROSA NICD mouse line 35 .Following tamoxifen-mediated induction, MuSCs are both null for Kmt5a and overexpress the Notch intracellular domain (NICD), a DNA-binding domain that promotes the expression of Notch target genes.NICD overexpression rescued the expression of key Notch target genes such as Rbpj and Hes1 without altering Kmt5a expression (Figure S6C).Restoring Notch signaling in Kmt5a KO MuSCs prevented their loss and decreased the number of Ki67+ cells, suggesting that it preserved quiescence (Figure 3H-J).However, skeletal muscle regeneration was still severely impaired compared to that in wild-type mice, analogous to that observed in Kmt5a KO mice, and we did not detect surviving MuSC at 21dpi (Figure S6D, E).To further assess the fate of MuSCs derived from double-mutant mice, we cultured freshly isolated cells and assessed their survival.Similar to the single mutant Kmt5a KO cells, the double mutant cells displayed a decline in cell survival after 72 h of culture (Figure S6F).These results indicate that, while Kmt5a epigenetically controls MuSC quiescence maintenance through Notch signaling, subsequent cell survival after quiescence exit involves a distinct mechanism.

Kmt5a safeguards MuSCs from ferroptosis.
We took advantage of our transcriptomic data to further assess the cell fate and determine the fate of the subpopulation of Kmt5a KO MuSCs that exit quiescence.Curated enrichment analysis for signi cantly modi ed genes highlighted ferroptosis in both the RNA-and PRO-seq datasets, whereas the pausing index was enriched for Notch signaling and several cancer-related pathways, consistent with the observations in Figure 3 (Figure S7A-E).Ferroptosis is a unique form of programmed cell death regulated by iron-mediated increases in reactive oxygen species that lead to lipid peroxidation, ultimately resulting in membrane rupture, cell death, and release of pro-in ammatory factors 36 .Mechanistically, this process is not completely understood and is the focus of extensive investigations for its potential contribution to cancer, aging, and frailty 37,38 .Owing to recent advances in this eld, ferroptosis is associated with several molecular, morphological, and biochemical hallmarks of ferroptosis 39 .The transcriptional signature in Kmt5a-null MuSCs suggested a pro-ferroptotic fate with a pathway activation z-score of 4.15 and an enrichment score of 44.06 (Adj.p value = 0.02650) (Figure S7A).We did not nd a strong correlation between ferroptosis markers and the pausing index (Figure S7F), suggesting that ferroptosis might not be regulated through promoter-proximal pausing.Interestingly, most of the genes silenced in Kmt5a KO MuSCs were anti-ferroptotic and either involved in iron processing and export (Ftl1 and Pcbp2) or inhibition of lipid peroxidation and cellular damage (Gclc, Gss, and Gpx4).In contrast, the upregulated genes were either markers of ferroptosis (Ptgs2, Cybb, and Hmox1) or genes that promoted intracellular iron import (Tfrc) (Figure S7F).
Based on these results, we hypothesized that Kmt5a-de cient MuSCs would exhibit impaired iron metabolism, aberrant accumulation of intracellular iron, and increased levels of lipid peroxidation, resulting in cell death by ferroptosis (Figure S7G).To test this hypothesis more directly, we rst characterized MuSC sensitivity to ferroptosis using gold standard compounds to induce ferroptosis (erastin and RSL3), along with Ferrostatin-1 (Fer1), which traps lipid radicals to rescue the effects of erastin and RSL3 (Figure S8) 36 .Both erastin and RSL3 induced a canonical ferroptotic response, with decreased cell viability, increased ROS production, and enhanced lipid peroxidation, which were rescued by Fer1 treatment (Figure S8).MuSCs were more sensitive to RSL3 exposure, perhaps because they directly target GPX4, which plays a critical role in preventing lipid peroxidation, and thus provides a major safeguard against ferroptosis.Interestingly, Gpx4 was among the most repressed genes identi ed in our scRNA-seq analysis (Figure S5B), particularly in the population of Kmt5a-de cient MuSCs that exhibited a cell cycle re-entry molecular signature.In vivo, we found that GPX4 was expressed in tibialis anterior muscle bers with a small cross-sectional area, implying a potential role for GPX4 in type 2A bers 40 (Figure 4A).Interestingly, Kmt5a KO MuSCs were virtually devoid of GPX4 and exhibited iron-rich pockets in the vicinity of the MuSC niche (Figure 4B and 4C).Less than 2% of MuSCs from wild-type mice exhibited detectable levels of intracellular iron (Fe 2+ ), whereas over 80% of Kmt5a KO MuSCs exhibited accumulation of iron foci (Figure 4C).Electron microscopy further suggested that Kmt5a KO MuSCs displayed hallmarks of ferroptosis, including cell swelling, plasma membrane blebbing and rupture (blue arrows), increased mitochondrial content, and intracellular iron accumulation (black arrows) (Figure 4D).
ICP-MS was used to measure the total iron content in freshly sorted MuSCs.Kmt5a KO MuSCs had ~3.4x10 -2 ng of iron per cell, which was nearly 70-fold higher than that measured in wild-type MuSCs (Figure 4E).Consistent with these ndings, the Kmt5a KO MuSCs exhibited higher levels of lipid peroxidation (Figure 4F), which progressively increased over time (Figure 4G).Likewise, genetic Kmt5a deletion resulted in the progressive rise of a pro-ferroptotic transcriptomic signature in Kmt5a KO MuSCs, including repression of Gpx4 and Rgs4 and higher expression of Ptgs2 41,42 (Figure 4H).
Next, we wondered if Kmt5a catalytic inhibition could promote ferroptosis.Kmt5a inhibition was accompanied by signs of ferroptosis at both the cellular and molecular levels.Dosage under the inhibitor IC 50 ([C] = 4uM < IC 50 = 7.3uM), while still signi cantly decreasing H4K20me1, was seemingly lethal in under 24h (Figure 4I, 4J, S8J and S8K).Loss of H4K20me1 following treatment was accompanied by decreased GPX4 protein and mRNA levels, as well as repression of Rgs4 and increased Ptgs2 and Hmox1 levels, mirroring the effects observed in RSL3 treated cells (Figure 4I-K and S8H-L).Kmt5a inhibition also signi cantly increased lipid peroxidation, which was reduced when co-treated with Fer-1 (Figure 4L and  4M), further supporting that cell death promoted by Kmt5a inhibition is ferroptosis.
Taken together, these results suggest that Kmt5a activity prevents premature ferroptotic MuSC death by epigenetically regulating key genes involved in iron metabolism and antioxidative activity upon cell cycle re-entry.

Epigenetic erosion of H4K20 drives MuSC population drift toward ferroptosis during aging.
Although not previously molecularly linked, increased pro-in ammatory features, loss of MuSC quiescence, and premature death are the hallmarks of skeletal muscle aging.While ferroptosis is an incompletely understood form of programmed cell death, especially in stem cell homeostasis, recent evidence indicates that ferroptosis is implicated in the pathogenesis of various age-dependent disorders 38 .Ferroptosis shares most, if not all, of the hallmarks of aging.Here, we aimed to con rm whether the key ndings observed in Kmt5a KO MuSCs are physiologically relevant to aging.Speci cally, we assessed the contribution of Kmt5a and H4K20me1 to the age-dependent decline in MuSC number and function.Since we found that aged MuSCs displayed lower levels of both H4K20me1 and Kmt5a than MuSCs from young mice (Figure 1G-M), we used ow cytometry to assess the activation status of aged MuSCs upon loss of H4K20me1 at the single-cell level.Flow cytometry analyses revealed that while virtually all quiescent MuSCs from young mice exhibited high levels of H4K20me1 (Ki67-H4K20me1 high ), an age-dependent reduction in this population of MuSCs suggests H4K20me1 erosion (Figure 5a), consistent with the information theory of aging 4 .Furthermore, more than 95% of MuSCs positive for the cell cycle entry marker Ki67 showed lower levels of H4K20me1, consistent with decreased H4K20 methylation being necessary for quiescence exit, and thus a hallmark of MuSC early activation (Pax7 + Ki67 + H4K20me1 low ) (Figure S9A).
To further understand how Kmt5a epigenetically controls MuSC fate, we mapped H4K20me1 in MuSCs from young and aged mice using CUT&Tag (Figure S9B).Consistent with previous studies using somatic cells, we found that H4K20me1 is a broad epigenetic mark with narrow peaks mostly localized near the TSS of genes and broader peaks spanning over the body of expressed genes (Figure 5A, B, and Figure S9C).To gain further insights into H4K20me1 regulation, we integrated H4K20me1 maps with our RNAseq data and found that H4K20me1 was associated with transcriptional status regardless of age (Figure S9D).Term analysis revealed that genes with a loss of H4K20me1 at their TSS were associated with the cell cycle and chromatin organization, while genes that gained H4K20me1 signal were notably enriched for the activation of cell death (Figure S9E).Further, GSEA identi ed key processes that were also identi ed in Kmt5a KO MuSCs, including Notch signaling, P53 signaling, and myogenesis (FDR<0.1)(Figure S9F-H).Most notably, we con rmed that Kmt5a acts as a master epigenetic regulator of Notch Signaling, as over 78% of Notch genes were repressed in aged MuSCs in an H4K20me1-dependent manner with a Pearson correlation p-value of 0.0023 (Figure 5C; bold).Although Notch relies on signaling-based gene regulation, ferroptosis is primarily a metabolic process.Therefore, the role of H4K20me1 in the transcriptional regulation of ferroptotic genes was unclear, except for a few key genes, notably Gpx4, which exhibited the most signi cant reduction in transcript levels (Figure 5D, bold).Instead, these data highlight that aged MuSCs display signs of a pro-ferroptotic transcriptional signature with repressed Glutathione Metabolism genes such as Gclm and Gclc, enhanced iron import, and handling with elevated iron-responsible element genes such as Tfrc, Fth1, and Ftl1, as well as canonical markers of ferroptosis, such as high Ncoa4, Hmox1, and Ptgs2.
To further characterize the possible presence of a pro-ferroptosis MuSC population during aging, we performed high-depth scRNA-seq on MuSCs from adult and aged mice (Figure 6A-C).Enrichment analysis for the most altered genes highlighted Ribosome as the top pathway, indicative of the high transcriptional and translational turnover associated with MuSC activation.We identi ed Ferroptosis and Glutathione Metabolism as among the pathways most enriched in MuSCs from aged mice (Figure 6D).Pseudotime analysis identi ed a novel subpopulation of aged MuSCs (Cluster 3) displaying hallmarks of ferroptosis such as low levels of Gpx4, Slc7a11, and Pcbp2, and high Ftl1 and Fth1 (Figure 6C-E).Both aging clusters (2 and 3) displayed similar aging signatures, such as decreased quiescence gene expression (Pax7, Spry1, and Rbpj).However, unlike the canonical aging cluster 2 43,44 , the ferroptotic cluster (cluster 3) differentiated itself by displaying no signs of premature commitment (MyoD and MyoG) or premature senescence (Cdkn2a and Trp53).Interestingly, Kmt5a was also signi cantly downregulated along with Gpx4, mostly in cluster 3, suggesting that the loss of Kmt5a could directly contribute to enhanced ferrosensitivity (Figure 5E and Figure S9I, J).
To better understand the extent to which MuSC ferroptosis contributes to skeletal muscle aging, we performed ow cytometry on adult and aged MuSCs, and assessed their viability, cell fate, and different forms of programmed cell death 12,43 .As previously shown, only a small percentage of aged MuSCs entered senescence prematurely (as opposed to geriatric MuSCs) 43 (Figure 5F; SPiDER + ) 11 .The remaining and the majority of dying aged MuSCs were skewed toward ferroptosis (~42%; AnnexinV -; Lipid Peroxidation High ), with fewer cells undergoing apoptosis (~27%; AnnexinV + ), indicating that ferroptosis is the dominant mode of regulated cell death in aged MuSCs (Figure 6F).Upon sorting, ferroptotic aged MuSCs expressed very low levels of Gpx4 and Kmt5a, but high levels of Hmox1 and Ptgs2, consistent with a canonical pro-ferroptotic fate response and similar to cluster 3 (Figure 6E and G).Upon assessing ferroptosis in vivo, we found that <10% of aged MuSCs displayed faint levels of cytoplasmic GPX4 in concert with an abundance of intracellular iron (Figure 6H and Figure S9K), which was not observed in MuSCs from young mice.This suggests that while aged MuSCs are defective in processing iron and fail to resist ferroptosis, some aged MuSCs retain the capacity to express GPX4, thus suggesting a reversible process.Similar to Kmt5a KO MuSCs, a signi cant proportion of aged MuSCs displayed high intracellular iron levels (>31%), with a concentration far exceeding that measured in MuSCs from young mice (Figure 6H, I).Interestingly, labile iron accumulation has also been observed in aged hematopoietic stem cells 45 .
While circulating iron is largely depleted with age (anemia), labile iron is often enriched in the aging tissues.Iron enrichment in aged skeletal muscles is believed to contribute to both functional and regeneration impairments as well as forms of muscle wasting, such as sarcopenia 37 .Consistent with reports showing that increased intramuscular iron is associated with increased lipid peroxidation 46 , we found that freshly isolated aged MuSCs displayed a 1.5-fold increase in lipid peroxidation during homeostasis (Figure 6J).To avoid stress response bias, we used MuSCs from Rosa CreERT2 ;Nrf2 f/f ;Gclc f/f mice 47,48 treated with tamoxifen (50 mg/kg i.p. for ve days), which produces Nrf2-Gclc double-KO cells, as a control, since a lack of glutathione in these cells naturally increases ROS production and lipid peroxidation 49 .Notably, Glutathione Metabolism was also enriched in aged MuSCs (Figure 6D), consistent with altered glutathione metabolism, which is a hallmark of stem cell ageing 50 .
Upon plating, the rate of lipid peroxidation dramatically increased (>4.5-fold increase) in aged MuSCs compared to that in younger cells, highlighting the enhanced sensitivity of aged MuSCs to oxidative and replicative stress outside their niche (Figure 6K).Although RSL3 treatment increased lipid peroxidation to comparable levels in MuSCs from both young and aged mice, Fer1 co-treatment rescued these effects.Importantly, Fer1 treatment alone prevented the increase in basal levels of lipid peroxidation observed in aged MuSCs (Figure 6K) and signi cantly enhanced both their viability and myogenic potential (Figure 6L-O).Taken together, these results demonstrate that ferroptosis is a dominant contributor to the decline in MuSC numbers with age, where activation of the ferroptotic program is triggered by the loss of Kmt5a.
Lastly, because human hematopoietic stem cells were recently shown to be selectively vulnerable to ferroptosis 51 and labile iron accumulation was observed in aged hematopoietic stem cells 45 , we wondered whether age-associated death by ferroptosis is a shared fate across other adult stem cells.As previously described, we found that both short-and long-term HSCs were enriched in the aged bone marrow (Figure S10 A, B), with long-term HSCs displaying myeloid-bias CD41, a hallmark of HSC aging (Figure S10C).Upon sorting long-term HSCs from young and aged mice, we found that only aged LT-HSCs displayed enhanced lipid peroxidation (Figure S10D).
Taken together, these data indicate that lipid peroxidation and cell death by ferroptosis are important mediators of stem cell aging, and warrant further exploration.
Prevention of systemic in ammation averts muscle stem cell aging.
Finally, because we found that Kmt5a levels decreased in MuSCs in response to both distal injury and ectopic induction of acute systemic in ammation, we assessed whether long-term inhibition of these proin ammatory molecules could restore Kmt5a levels in MuSCs, prevent Kmt5a-mediated loss of quiescence and ferroptotic cell death, and improve skeletal muscle homeostasis and regeneration during aging (Figure 7).First, we con rmed that aged mice treated with an anti-in ammatory drug speci cally targeting our pathway of interest (Bindarit 1/wk IP 30 mg/kg [12to24-30mo]) exhibited signi cantly diminished cellular and molecular in ammatory pro les similar to those observed in young mice (Figure S11A-K).As expected, Bindarit treatment restored Kmt5a levels in MuSCs from aged mice back to young levels (Figure S11L).In vivo, the number of MuSCs was signi cantly improved both at homeostasis and after regeneration compared to vehicle-treated aged mice (Figure 7A-D), complemented by enhanced muscle repair and restoration of muscle strength after injury (Figure 7D-G).Finally, both intracellular iron and lipid peroxidation signi cantly decreased in MuSCs derived from Bindarit-treated aged mice (Figure 7H and I).Unexpectedly, we observed that Bindarit-treated mice were leaner (Figure S11M) and had an extended lifespan (Figure S11N).Improvement in muscle regeneration was not observed in adult mice, despite the presence of fewer circulatory monocytes (Figure S12A-F).It was also not detrimental, possibly bene ting from circumstances with chronic in ammation only, as previously reported [52][53][54] .

Discussion
Our study provides insight into the interplay between in ammation, epigenetics, and stem cell fate in the context of aging.Through the use of genetic models and multi-omics analysis, we propose a novel mechanism whereby the decline of Kmt5a and its associated histone mark H4K20me1 in aged muscle stem/progenitor cells (MuSCs) triggers premature exit from quiescence, ultimately resulting in cell death by ferroptosis.Genetic deletion of Kmt5a causes disruption of quiescence and loss of MuSC numbers over time.The loss of the Kmt5a-mediated histone mark H4K20me1 in MuSCs led to large transcriptional changes, including Notch target gene silencing, which functionally leads to quiescence exit.Upon activation, MuSCs lacking the ability for de novo H4K20me1 deposition prematurely die through ferroptosis, a yet uncharacterized form of regulated cell death in MuSCs.Yet, ferroptosis was identi ed as the dominant form of regulated cell death in aged MuSCs.The roles of ferroptosis and iron metabolism in MuSCs, especially in aging, are currently unclear.Iron metabolism and ferroptosis contribute to muscular pathogenesis, such as sarcopenia and rhabdomyosarcoma, which have recently gained traction but remain largely unknown 44 .MuSC-speci c deletion of Tfrc, a receptor that mediates the intracellular import of transferrin-bound iron, impairs skeletal muscle regeneration by causing irreversible depletion of the pool and further cell-autonomous defects in proliferation and differentiation 46,55 .Surprisingly, the study showed that preventing Tfrc-mediated import of iron in MuSCs leads to labile iron accumulation and lipid peroxidation in the regenerating muscles, seemingly inducing ferroptosis within the regenerating muscle.Taken together, these observations suggest that not only iron is an important component of myogenesis 56 , but iron scavenging by MuSC-derived myoblasts and newly generated bers is important for preventing aberrant lipid peroxidation and ROS damage in the niche during skeletal muscle regeneration.Although the precise contribution of MuSC iron homeostasis upon exit from quiescence remains to be explored, the role of iron in DNA replication and the G1 to S-phase transition has been suggested previously [57][58][59] , consistent with the inability of Kmt5a KO MuSCs to progress through the cell cycle, as we found very little EdU incorporation in Ki67+ Kmt5a KO MuSCs.The regulation of iron metabolism, similar to skeletal muscle regeneration, requires complex interactions among various cell types.Therefore, further investigation is required to determine how changes in iron handling affect skeletal muscle aging and resident cell functions.
Interestingly, a recent study has highlighted that MuSCs maintain these changes for weeks after acute skeletal muscle injury 23 .However, the exact mechanism by which MuSCs sustain this enhanced regenerative ability long after the injury has healed, especially when speci c systemic factors have receded from the circulatory system, remains unclear.The concept of epigenetic memory provides a plausible explanation of this phenomenon.In the context of adaptive immunity against in ammation and vaccination, this phenomenon illustrates how in ammatory signals can direct long-term memory immunity via intertwined epigenetic and metabolic shifts 63 .Such epigenetic mechanisms allow cells to retain speci c transcriptional patterns for extended periods and even transmit these features to progenies 25 .Importantly, Yang et al. recently demonstrated that the loss of epigenetic information is not merely a symptom but also a uni ed causative agent of aging across mammals 4 , closely intertwined with rising systemic in ammation 64 .However, the aged epigenetic landscape of quiescent stem cells, including MuSCs, remains largely uncharted 7,65,66 , especially regarding the in uence of age-related systemic in ammation on the aged MuSC epigenome and transcriptome.Here, we found that several proin ammatory cytokines are increased in the aged mouse plasma and are associated with CCR2 signaling activation in aged skeletal muscles.To delineate the role of these cytokines in MuSC aging, we administered a cocktail of chemokines to adult mice and assessed MuSC function.Systemic exposure to CCR2-ligands is su cient to repress Kmt5a in adult MuSCs, causing long-term loss of H4K20me1.In Kmt5a KO MuSCs, or in wild-type cells exposed to systemic CCR2-ligands, the decrease in H4K20me1 was accompanied by G alert features such as swifter cell cycle re-entry, accelerated myogenesis, and enhanced regeneration.Taken together, these cellular and molecular shifts indicated a prolonged injury response shaped by transient in ammatory signals.However, chronic exposure to CCR2-ligands was su cient to decrease Kmt5a and H4K20me1 levels and trigger premature cell death via ferroptosis in MuSCs, partially recapitulating the effects of Kmt5a deletion.During regeneration, an acute immune response followed by its resolution is critical for the spatiotemporal progression of myogenesis and successful regeneration 67 .In contrast, chronic in ammation is often implicated in aging and other related disorders.Considering the physiological role of Kmt5a-mediated epigenetic remodelling in response to acute in ammation, we postulated that it serves as an epigenetic switch to prime MuSCs into G alert by silencing Notch target genes.However, sustained in ammation, as observed in aging, could permanently repress Kmt5a.This could irreversibly prevent H4K20me1 de novo deposition, leading to the detrimental effects observed in Kmt5a-null MuSCs.Thus, we propose that systemic in ammation drives MuSC aging through epigenetic erosion.While these observations are compelling, comprehensive studies are vital to determine whether this phenomenon extends to other aging stem cells 68 .Kmt5a-mediated deposition of H4K20me1 is critical for mouse development, and deletion of Kmt5a results in embryonic lethality before the eight-cell stage 69 .Interestingly, the speci c deletion of Kmt5a in MuSCs did not result in immediate depletion of the pool.Instead, it mirrored an accelerated stochastic model of aging, whereby MuSC decline is a progressive albeit exponential phenomenon.We hypothesized that the stochastic nature of MuSC decline might correspond to the loss of H4K20me1 over time following Kmt5a deletion.Both Kmt5a and H4K20me1 are highly regulated in a cell cycle-dependent manner, enabling proper DNA replication during S phase and safeguarding genomic integrity during mitosis 70 .In somatic cells, Kmt5a deletion result in genomic instability and rapid cell death.Since we detected a signi cant proportion of Ki67+ Kmt5a KO MuSCs, and that most of the Kmt5a KO MuSCs remaining cells were H4K20me1+ six-weeks after deletion, it is possible that mutant MuSCs can survive out of quiescence for as long as they have H4K20me1, supporting the assumption that Kmt5a regulates quiescence and survival via two distinct mechanisms.Further supporting this idea, reactivating Notch signaling with the NICD model prevented the depletion of the Kmt5a KO MuSCs pool at homeostasis, but not following injury.Although restoring Notch could not prevent ferroptosis in Kmt5a KO MuSCs, it is also possible that the overactivation of NICD leads to Kmt5a-independent myogenic impairment 35 .While our data suggest that the loss of H4K20me1, rather than Kmt5a, results in MuSC exit from quiescence and subsequent ferroptosis, further investigation is required to determine what mediates the loss of H4K20me1 following Kmt5a deletion.In addition, because Kmt5a-mediated maintenance of monomethylation is required for the subsequent deposition of di-and trimethylation of H4K20, dissociation of the exact contribution of Kmt5a and H4K20me1 requires further investigation.The scarcity of adult stem cells combined with the eeting status of quiescence makes the study of H4K20 methylation dynamics a challenge that will require both collaborative efforts and the use of new technologies.
Our ndings propose a novel mechanistic axis of stem cell aging that links in ammation to Kmt5adependent epigenetic regulation of stem cell metabolism, which extends recent ndings.Benjamin et al, recently showed that a subset of aged MuSCs is dysfunctional due to high NF-κB-mediated inhibition of NRF2, thus leading to unbalanced glutathione metabolism, which is critical for limiting lipid peroxidation and ferroptosis 49,50 .Given that Bindarit preferentially targets the NF-κB pathways 71 , the observed bene cial effects in Bindarit-treated aged mice are in line with improved glutathione metabolism.Additionally, increased in ammation and iron accumulation are closely associated with human aging.
Age-related chronic in ammation leads to increased iron accumulation in various tissues, whereas excess iron promotes in ammation through the generation of reactive oxygen species and activation of pro-in ammatory signaling pathways 38 .This reciprocal relationship between in ammation and iron levels is thought to contribute to the development and progression of age-related diseases.Our data suggest that in ammation may induce epigenetic changes that lead to iron accumulation and potential ferroptotic death in stem cells, which could have important implications for human aging.Because chronic in ammation is a hallmark of aging, it is plausible that the accumulation of iron in stem cells due to in ammation-induced epigenetic reprogramming contributes to age-associated low-grade in ammation.The release of pro-in ammatory molecules associated with ferroptotic death can perpetuate the in ammatory environment, creating a vicious cycle of in ammation, iron accumulation, and ferroptotic death across several aged tissues.
In this study, we present data indicating that age-associated systemic in ammation reprograms MuSCs through epigenetic erosion, leading to the premature loss of quiescence and subsequent cell death by ferroptosis.Our ndings directly link aging and in ammation to the loss of Kmt5a function that drives an epigenetic switch to disrupt MuSC quiescence and promote ferroptosis.Our ndings provide evidence to suggest that the in ammation-epigenetic-ferroptosis axis has signi cant therapeutic implications in stem cell rejuvenation.Although iron metabolism is known to contribute to stem cell aging 45 , our study sheds light on this mechanism and highlights its importance in developing new therapeutic avenues.Encouragingly, iron chelators, which are already used for other diseases, can be rapidly translated into stem cell therapy in elderly patients.In addition to emerging evidence on the involvement of ferroptosis in aging disorders, particularly neurodegenerative diseases 72,73 , we present a compelling case for further exploration of the complex interplay between epigenetic erosion-mediated diversity, iron metabolism, and ferroptosis in diverse adult stem cell compartments during aging.Our ndings underscore the urgent need for regenerative medicine to further explore the fundamental mechanisms driving these processes with the aim of developing novel therapeutic interventions to combat age-related degenerative diseases.Unraveling these mysteries may pave the way for a new era of personalized regenerative medicine that signi cantly improves the quality of life of aging individuals and potentially extends human lifespan.

Limitations of study
Although our study sheds light on the intricate mechanisms linking in ammation, epigenetics, and ferroptosis in MuSC aging, there are several limitations that warrant consideration.First, the aging experiments were restricted to male mice.We also focused on speci c genetic strains and a particular age bracket, potentially overlooking the nuances present in different genetic backgrounds and across a broader age spectrum.Further experiments are required to determine whether these mechanisms are conserved in humans.Additionally, our study predominantly emphasized the role of Kmt5a and H4K20me1, and the involvement of other epigenetic marks cannot be ruled out, especially regarding the H4K20me1 loss effects on H4K20me2 and me3.While we believe that the loss of H4K20me1 in MuSCs contributes to quiescence exit, the initiating mechanisms that trigger H4K20me1 erosion during quiescence remain unclear.Furthermore, the complex interactions between various cell types in the regulation of iron metabolism and skeletal muscle regeneration necessitate a more exhaustive exploration.Lastly, while we observed systemic bene cial effects of Bindarit on aged mice, the exact underlying mechanisms of this rejuvenation remain to be fully explored.

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